WO2021203042A1 - Approche d'immunothérapie adoptive universelle pour traiter la covid-19 et des maladies infectieuses émergentes futures - Google Patents

Approche d'immunothérapie adoptive universelle pour traiter la covid-19 et des maladies infectieuses émergentes futures Download PDF

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WO2021203042A1
WO2021203042A1 PCT/US2021/025638 US2021025638W WO2021203042A1 WO 2021203042 A1 WO2021203042 A1 WO 2021203042A1 US 2021025638 W US2021025638 W US 2021025638W WO 2021203042 A1 WO2021203042 A1 WO 2021203042A1
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cells
coronaviridae
virus
composition
antigen
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PCT/US2021/025638
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Francois Binette
Brian M. CULLEY
Rami Skaliter
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Lineage Cell Therapeutics, Inc.
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Priority to US17/995,008 priority Critical patent/US20230210978A1/en
Publication of WO2021203042A1 publication Critical patent/WO2021203042A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A61K39/215—Coronaviridae, e.g. avian infectious bronchitis virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4615—Dendritic cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/462—Cellular immunotherapy characterized by the effect or the function of the cells
    • A61K39/4622—Antigen presenting cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A61K39/464454—Enzymes
    • A61K39/464457—Telomerase or [telomerase reverse transcriptase [TERT]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/464838—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/16—Antivirals for RNA viruses for influenza or rhinoviruses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0639—Dendritic cells, e.g. Langherhans cells in the epidermis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/515—Animal cells
    • A61K2039/5154—Antigen presenting cells [APCs], e.g. dendritic cells or macrophages
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/515—Animal cells
    • A61K2039/5156—Animal cells expressing foreign proteins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
    • C12N2506/03—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from non-embryonic pluripotent stem cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2510/00—Genetically modified cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/20011—Coronaviridae
    • C12N2770/20034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • Coronaviruses belong to the family Coronaviridae and are enveloped, positive-sense, single- stranded RNA viruses.
  • the coronavirus genome is approximately 31 kb in size, making these viruses the largest known RNA viruses yet identified.
  • Coronaviruses infect a variety of hosts including humans and several other vertebrates. Coronaviruses are associated with several respiratory and intestinal tract infections. Respiratory coronaviruses have long been recognized as significant pathogens in domestic and companion animals and as the cause of upper respiratory tract infections in humans.
  • HCVs human coronaviruses
  • HCoVs human coronaviruses
  • the common cold and croup e.g.: HCoV-229E, HCoV-OC43, HCoV-NL63 and HCoV-HKU
  • SARS-CoV-2 SARS-CoV-2, which causes COVID-19.
  • the dendritic cell (DC) vaccine platform described herein may address both by mobilizing immune systems to specifically target SARS-CoV-2 viral particles and infected cells, while large cell banks can be created to adapt quickly to future infectious diseases.
  • the present disclosure provides a method of making a composition for treating an infectious disease, said method comprising obtaining a cell line of human pluripotent stem (hPS) cells; differentiating said hPS cells into a population of mature antigen-presenting cells, wherein said antigen expressing cells express an antigen of a Coronaviridae virus; genetically altering said hPS cells before or after they are differentiated so that they express a protein comprising one or more immunogenic epitopes of said Coronaviridae virus; and formulating said differentiated cells to provide a composition for administration to a human subject.
  • hPS human pluripotent stem
  • the Coronaviridae virus is SARS-CoV-2.
  • the antigen-presenting cells are selected from the group consisting of dendritic cells, macrophages, B cells, and a combination thereof.
  • the composition is administered to a human for treating a disease caused by a Coronaviridae virus infection.
  • the composition is administered to a human for preventing a disease caused by a Coronaviridae virus infection.
  • the composition is a vaccine.
  • the Coronaviridae antigen comprises the Coronaviridae spike protein. In some embodiments, the Coronaviridae antigen comprises the full-length spike protein. In some embodiments, the Coronaviridae antigen comprises the spike protein receptor binding domain (RBD). In some embodiments, the Coronaviridae antigen comprises a 209 amino acid sequence of the RBD. In some embodiments, the Coronaviridae antigen comprises the spike protein SI domain, S2 domain, or both. In some embodiments, the Coronaviridae antigen comprises the nucleocapsid protein. In some embodiments, the Coronaviridae antigen comprises the membrane protein.
  • RBD spike protein receptor binding domain
  • the Coronaviridae antigen comprises a 209 amino acid sequence of the RBD.
  • the Coronaviridae antigen comprises the spike protein SI domain, S2 domain, or both.
  • the Coronaviridae antigen comprises the nucleocapsid protein. In some embodiments, the Coronaviridae antigen comprises the membrane protein.
  • genetically altering said hPS cells comprises transfecting the cells with a polynucleotide encoding a Coronaviridae virus antigen and the cytoplasmic tail of lysosomal associated membrane protein 1 (LAMP-1).
  • the polynucleotide further comprises a poly(A) tail and a gene which encodes heat shock protein 96 (HSP96).
  • the present disclosure provides a composition according to any one of the methods described herein.
  • the Coronaviridae virus is SARS-CoV-2.
  • the antigen-presenting cells are selected from the group consisting of dendritic cells, macrophages, B cells, and a combination thereof. In some embodiments, the antigen-presenting cells are dendritic cells.
  • the composition is administered to a human for treating a disease caused by a Coronaviridae virus infection.
  • the composition is administered to a human for preventing a disease caused by a Coronaviridae virus infection.
  • the Coronaviridae antigen comprises the Coronaviridae spike protein. In some embodiments, the Coronaviridae antigen comprises the full-length spike protein. In some embodiments, the Coronaviridae antigen comprises the spike protein receptor- binding domain (RBD). In some embodiments, the Coronaviridae antigen comprises a 209 amino acid sequence of the RBD. In some embodiments, the Coronaviridae antigen comprises the spike protein SI domain, S2 domain, or both. In some embodiments, the Coronaviridae antigen comprises the nucleocapsid protein. In some embodiments, the Coronaviridae antigen comprises the membrane protein.
  • RBD spike protein receptor- binding domain
  • the Coronaviridae antigen comprises a 209 amino acid sequence of the RBD.
  • the Coronaviridae antigen comprises the spike protein SI domain, S2 domain, or both.
  • the Coronaviridae antigen comprises the nucleocapsid protein. In some embodiments, the Coronaviridae antigen comprises the membrane protein
  • genetically altering said hPS cells comprises transfecting the cells with a polynucleotide encoding a Coronaviridae virus antigen and the cytoplasmic tail of lysosomal associated membrane protein 1 (LAMP-1).
  • the polynucleotide further comprises a poly(A) tail and a gene which encodes heat shock protein 96 (HSP96).
  • the composition is a vaccine.
  • the present disclosure provides a composition comprising dendritic cells expressing a Coronoviridiae antigen.
  • the Coronaviridae antigen comprises the Coronaviridae spike protein. In some embodiments, the Coronaviridae antigen comprises the full-length spike protein. In some embodiments, the Coronaviridae antigen comprises the spike protein receptor- binding domain (RBD). In some embodiments, the Coronaviridae antigen comprises a 209 amino acid sequence of the RBD. In some embodiments, the Coronaviridae antigen comprises the spike protein SI domain, S2 domain, or both. In some embodiments, the Coronaviridae antigen comprises the Coronaviridae nucleocapsid protein. In some embodiments, the Coronaviridae antigen comprises the Coronaviridae membrane protein.
  • RBD spike protein receptor- binding domain
  • the Coronaviridae antigen comprises a 209 amino acid sequence of the RBD.
  • the Coronaviridae antigen comprises the spike protein SI domain, S2 domain, or both.
  • the Coronaviridae antigen comprises the Coronaviridae nucleocapsid protein. In some
  • genetically altering said hPS cells comprises transfecting the cells with a polynucleotide encoding a Coronaviridae virus antigen and the cytoplasmic tail of lysosomal associated membrane protein 1 (LAMP-1).
  • the polynucleotide further comprises a poly(A) tail and a gene which encodes heat shock protein 96 (HSP96).
  • FIG. 1 schematic describing the Dendritic Cell Infectious Diseases Program.
  • FIG. 2 - graph describing the features and results derived from dendritic cell vaccines.
  • FIG. 3 - shows relevant non-clinical data obtained with dendritic cell vaccines.
  • FIG. 4 - provides approaches for scaling up production of vaccines.
  • FIG. 5 - shows flow cytometry graphs for the various groups studied in Example 3.
  • FIG. 6 - shows flow cytometry data for efficacy of incorporation of spike antigen into transfected cells by electroporation.
  • FIG. 7 - shows flow cytometry data for % incorporation of spike antigen versus % expression of CD86.
  • FIG. 8 shows flow cytometry data for % HLA-DR expression versus % expression of CD83.
  • FIG. 9 restriction map showing the pKAN- SARS-CoV-2 (COVID 19)-LAMP1 construct with a Kanamycin resistance gene.
  • FIG. 10 schematic diagram of the plasmid used to produce the SARS-CoV-2 (COVID 19) spike protein/LAMP-1 mRNA.
  • FIG. 11 denaturing Agarose gel showing size of the mRNA synthesized from the SARS-CoV-2 (COVID 19)-LAMP1 plasmid construct.
  • FlashGelTMRNA marker (ladder) was loaded on lane 1 and lane 12. FlashGelTMRNA marker consists of RNA transcripts 0.5kb-9kb. Lanes 2-11 show mRNA loaded at different concentrations as shown in the right-hand side of the image. Lane 13 is a no template control (negative control).
  • FIG. 12 schematic diagram of the SARS-CoV-2S LAMP-1 mRNA construct.
  • Cell culture refers to a plurality of cells grown in vitro over time.
  • the cell culture may originate from a plurality of hPS cells or from a single hPS cell and may include all of the progeny of the originating cell or cells, regardless of 1) the number of passages or divisions the cell culture undergoes over the lifetime of the culture; and 2) any changes in phenotype to one or more cells within the culture over the lifetime of the culture (e.g. resulting from differentiation of one or more hPS cells in the culture).
  • a cell culture begins with the initial seeding of one or more suitable vessels with at least one hPS cell and ends when the last surviving progeny of the original founder(s) is harvested or dies. Seeding of one or more additional culture vessels with progeny of the original founder cells is also considered to be a part of the original cell culture.
  • Coronaviruses belong to the family Coronaviridae and are enveloped, positive-sense, single- stranded RNA viruses.
  • the coronavirus genome is approximately 31 kb in size, making these viruses the largest known RNA viruses yet identified.
  • Coronaviruses infect a variety of hosts including humans and several other vertebrates. Coronaviruses are associated with several respiratory and intestinal tract infections. Respiratory coronaviruses have long been recognized as significant pathogens in domestic and companion animals and as the cause of upper respiratory tract infections in humans.
  • HCVs human coronaviruses
  • HCoVs human coronaviruses
  • common cold and croup e.g.: HCoV-229E, HCoV-OC43, HCoV-NL63 and HCoV-HKU.
  • Human coronaviruses such as SARS-CoV and MERS-CoV are also associated with severe respiratory illness.
  • Coronaviruses that induce respiratory tract disease in other vertebrate animals include mouse hepatitis virus- 1 (MHV-1) a natural mouse pathogen, infectious bronchitis virus (IBV) in chickens and other avian species, bovine coronavirus (BCoV) in cows and other ruminants, porcine respiratory syndrome virus (PRCV) in pigs and canine respiratory coronavirus (CRCoV) in dogs to name a few. (See, for example, Refs. 1-11).
  • MHV-1 mouse hepatitis virus- 1
  • IBV infectious bronchitis virus
  • BCoV bovine coronavirus
  • PRCV porcine respiratory syndrome virus
  • CRCoV canine respiratory coronavirus
  • DCs Dendritic cells
  • accessory cells also known as accessory cells
  • Their main function is to process antigen material and present it on the cell surface to the T cells of the immune system. They act as messengers between the innate and the adaptive immune systems.
  • treatment refers to the medical management of a subject with the intent to cure, ameliorate, stabilize, or prevent a disease, pathological condition, or disorder.
  • This term includes active treatment, that is, treatment directed specifically toward the improvement of a disease, pathological condition, or disorder, and also includes causal treatment, that is, treatment directed toward removal of the cause of the associated disease, pathological condition, or disorder.
  • this term includes palliative treatment, that is, treatment designed for the relief of symptoms rather than the curing of the disease, pathological condition, or disorder; preventative treatment, that is, treatment directed to minimizing or partially or completely inhibiting the development of the associated disease, pathological condition, or disorder; and supportive treatment, that is, treatment employed to supplement another specific therapy directed toward the improvement of the associated disease, pathological condition, or disorder.
  • palliative treatment that is, treatment designed for the relief of symptoms rather than the curing of the disease, pathological condition, or disorder
  • preventative treatment that is, treatment directed to minimizing or partially or completely inhibiting the development of the associated disease, pathological condition, or disorder
  • supportive treatment that is, treatment employed to supplement another specific therapy directed toward the improvement of the associated disease, pathological condition, or disorder.
  • subject includes, but is not limited to, animals, plants, bacteria, viruses, parasites and any other organism or entity.
  • the subject can be a vertebrate, more specifically a mammal (e.g., a human, horse, pig, rabbit, dog, sheep, goat, non-human primate, cow, cat, guinea pig or rodent), a fish, a bird or a reptile or an amphibian.
  • the subject can be an invertebrate, more specifically an arthropod (e.g., insects and crustaceans).
  • arthropod e.g., insects and crustaceans
  • the term does not denote a particular age or sex. Thus, adult and newborn subjects, as well as fetuses, whether male or female, are intended to be covered.
  • a patient refers to a subject afflicted with a disease or disorder.
  • patient includes human and veterinary subjects.
  • the compounds described herein can be administered to a subject comprising a human or an animal including, but not limited to, a mouse, dog, cat, horse, bovine or ovine and the like, that is in need of alleviation or amelioration from a recognized medical condition.
  • the subject is human.
  • the term “in need of treatment” as used herein refers to a judgment made by a caregiver (e.g. physician, nurse, nurse practitioner, or individual in the case of humans; veterinarian in the case of animals, including non-human mammals) that a subject requires or will benefit from treatment. This judgment is made based on a variety of factors that are in the realm of a care giver's expertise, but that include the knowledge that the subject is ill, or will be ill, as the result of a condition that is treatable by the disclosed compounds.
  • a caregiver e.g. physician, nurse, nurse practitioner, or individual in the case of humans; veterinarian in the case of animals, including non-human mammals
  • preventing refers to administering a compound prior to the onset of clinical symptoms of a disease or conditions so as to prevent a physical manifestation of aberrations associated with the disease or condition.
  • preventing refers to administering a compound prior to the onset of clinical symptoms of SARS-CoV-2 virus infection so as to prevent a physical manifestation of aberrations associated with SARS-CoV-2 virus infection.
  • feeder cells refers to non-hPS cells that are co-cultured with hPS cells and provide support for the hPS cells.
  • Support may include facilitating the growth and maintenance of the hPS cell culture by providing the hPS cell culture with one or more cell factors such that the hPS cells are maintained in a substantially undifferentiated state.
  • Feeder cells may either have a different genome than the hPS cells or the same genome as the hPS cells and may originate from a non-primate species, such as mouse, or may be of primate origin, e.g., human.
  • Examples of feeder cells may include cells having the phenotype of connective tissue such as murine fibroblast cells, human fibroblasts.
  • feeder-free refers to a condition where the referenced composition contains no added feeder cells.
  • feeder-free encompasses, inter alia, situations where primate pluripotent stem cells are passaged from a culture which may comprise some feeders into a culture without added feeders even if some of the feeders from the first culture are present in the second culture.
  • Serum free refers tissue culture growth conditions that have no added animal serum such fetal bovine serum, calf serum, horse serum, and no added commercially available serum replacement supplements such as B-27. Serum free includes, for example, media which may comprise human albumin, human transferrin and recombinant human insulin.
  • electroporation refers to a method for permeabilizing cell membranes by generating membrane pores with electrical stimulation.
  • the applications of electroporation include, but are not limited to, the delivery of DNA, RNA, siRNA, peptides, proteins, antibodies, drugs or other substances to a variety of cells such as mammalian cells, plant cells, yeasts, other eukaryotic cells, bacteria, other microorganisms, and cells from human patients.
  • a composition for treating an infectious disease including: a) obtaining a cell line of human pluripotent stem (hPS) cells; b) differentiating said hPS cells into a population of mature antigen-presenting cells, wherein said antigen expressing cells express an antigen of an infectious disease; c) genetically altering said hPS cells before or after they are differentiated so that they express a protein comprising one or more immunogenic epitopes of said infectious disease; and d) formulating said differentiated cells to provide a composition for administration to a human subject.
  • hPS human pluripotent stem
  • Pluripotent stem cells have the ability to both proliferate continuously in culture and, under appropriate growth conditions, differentiate into lineage restricted cell types representative of all three primary germ layers: endoderm, mesoderm and ectoderm (U.S. Patent Nos. 5,843,780; 6,200,806; 7,029,913; Shamblott et al., (1998) Proc. Natl. Acad. Sci. USA 95:13726; Takahashi et al., (2007) Cell 131(5):861; Yu et al., (2007) Science 318:5858).
  • a pluripotent stem cell will, under appropriate growth conditions, be able to form at least one cell type from each of the three primary germ layers: mesoderm, endoderm and ectoderm.
  • the PS cells may originate from pre-embryonic, embryonic or fetal tissue or mature differentiated cells.
  • an established PS cell line may be a suitable source of cells for practicing the invention.
  • the hPS cells are not derived from a malignant source.
  • hPS cells will form teratomas when implanted in an immuno-deficient mouse, e.g. a SCID mouse.
  • Prototype “human Pluripotent Stem cells” are pluripotent cells derived from pre-embryonic, embryonic, or fetal tissue at any time after fertilization, and have the characteristic of being capable under appropriate conditions of producing progeny of several different cell types that are derivatives of all of the three germinal layers (endoderm, mesoderm, and ectoderm), according to a standard art-accepted test, such as the ability to form a teratoma in 8-12 week old SCID mice.
  • hPS cells are not derived from a cancer cell or other malignant source. It is desirable (but not always necessary) that the cells be euploid.
  • Exemplary are embryonic stem cells and embryonic germ cells used as existing cell lines or established from primary embryonic tissue of human origin. This invention can also be practiced using pluripotent cells obtained from primary embryonic tissue, without first establishing an undifferentiated cell line.
  • hPS cells can be propagated continuously in culture, using culture conditions that promote proliferation while inhibiting differentiation.
  • ES cells are cultured on a layer of feeder cells, typically fibroblasts derived from embryonic or fetal tissue (Thomson et al., Science 282: 1145, 1998).
  • hPS cells can be maintained in an undifferentiated state even without feeder cells.
  • the environment for feeder-free cultures includes a suitable culture substrate, such as ECM-based hydrogel, such as solubilized basement membrane preparation extracted from the Engelbreth-Holm- Swarm (EHS) mouse sarcoma (e.g., MATRIGEL®), or laminin.
  • EHS Engelbreth-Holm- Swarm
  • MATRIGEL® solubilized basement membrane preparation extracted from the Engelbreth-Holm- Swarm
  • laminin laminin.
  • the cultures are supported by a nutrient medium containing factors that promote proliferation of the cells without differentiation (WO 99/20741).
  • Such factors may be introduced into the medium by culturing the medium with cells secreting such factors, such as irradiated primary mouse embryonic fibroblasts, telomerized mouse fibroblasts, or fibroblast-like cells derived from hPS cells (U.S. Patent 6,642,048).
  • Medium can be conditioned by plating the feeders in a serum free medium such as Knock-Out DMEM (Gibco), supplemented with serum replacement ranging from 10-30%, preferably 20% (US 2002/0076747 Al, Life Technologies Inc.) and 4 ng/mL bFGF.
  • a serum free medium such as Knock-Out DMEM (Gibco)
  • serum replacement ranging from 10-30%, preferably 20% (US 2002/0076747 Al, Life Technologies Inc.) and 4 ng/mL bFGF.
  • Some embodiments of the invention provide for maturing immature DC (imDC) to mature DC (mDC) by contacting the imDC with a suitable maturation cocktail comprising a plurality of exogenous cytokines.
  • the maturation cocktail may comprise GM-CSF.
  • Suitable maturation cocktails include any of the following: a) GM-CSF, TNFa, IL-lp, IFNy, and PGE2; b) GM-CSF, TNFa, IL-lp, IFNy, PGE2 and CD40L; c) GM-CSF, TNFa, IL-lp, IFNy, PGE2, POLY I:C, and IFNa; d) GM-CSF, TNFa, IL-lp, IFNy, POLY I:C, and IFNa; e) GM- CSF, TNFa, IL-lp, IFNy, POLY I:C, IFNa, and CD40L; f) TNFa, IL-lp, PGE2 and IL-6; g) GM- CSF, IL-lp, PGE2, and, IFNy; h) GM-CSF, TNFa, PGE2, and, IFNy; i) GM-CSF
  • ligands to one or more cytokine receptors may be used in place of, and/or in addition to the cytokine.
  • Other methods known in the art, may be used to mature imDC to mDC. Examples include contacting imDC with lipopolysaccharide (LPS), contacting the imDC with CpG containing oligonucleotides, injecting the imDC into an area of inflammation within a subject.
  • LPS lipopolysaccharide
  • the imDC may be cultured in the presence of the maturation cocktail, for at least about 12-15 hours, for at least about 1 day, for at least about 2 days, or for at least about 3 days to produce mDC. In some embodiments the imDC may be cultured in the presence of the maturation cocktail for about 24 hours to produce mDC. In other embodiments the imDC may be cultured in the presence of the maturation cocktail for about 48 hours to produce mDC.
  • mDC may express one or more markers such as CD83, CD86, MHCI and MHCII, but not CD 14 and may have functional properties similar to mature DC that are differentiated in vivo. Functional properties may include the capability to process and present antigen to an immunologically competent cell. Processing and presenting antigen may include for example the proteolysis of a target protein, as well as the expression and processing of a nucleic acid encoding a target antigen.
  • the mDC may also have the ability to migrate within peripheral and lymphoid tissue. Thus mDC differentiated from hPS cells according to the invention may be induced to migrate in response to an appropriate stimulus such as MIP3p.
  • the mDC may secrete one or more cytokines such as one or more pro-inflammatory cytokines. Exemplary cytokines secreted by DC according to the invention may include IL-12, IL-10 and IL-6.
  • the cells express one or more markers of CD86, CD83, or MHCII.
  • Various embodiments of the invention described herein provide methods of differentiating hPS cells into DCs. It is contemplated that the methods may further comprise mitotically inactivating various types of cells including unwanted hPS cells in a differentiated population as well as cells made according the methods described infra (e.g. any hematopoietic lineage cells, including mDC and imDC). Thus some embodiments of the invention may comprise contacting the DC cells with a protein or peptide antigen or a nucleic acid encoding an antigen and contacting the DC e.g. an mDC, with a radiation source or a chemical agent suitable for inhibiting cell division.
  • Exposure of the mDC to a radiation source or the chemical agent may be desirable where the mDC are contained in a population of cells comprising at least one hPS cell. Irradiating the cells or treating the cells with the chemical agent will inhibit cell division, while maintaining functionality of the mDC. Moreover, treating the cells with a radiation source or a chemical agent may minimize any undesirable effects stemming from the presence of hPS cells in the population.
  • the invention provides a method of differentiating hPS cells into mesoderm comprising contacting the hPS cells with a differentiation cocktail comprising a plurality of exogenous cytokines such as BMP-4, VEGF, SCF and optionally GM-CSF and culturing the cells for at least a day thereby differentiating hPS cells into mesoderm.
  • the cells may be cultured for at least about 2 days, at least about 3 days, at least about 4 days, at least about 5 days with the differentiation cocktail thereby differentiating the hPS cells into mesoderm.
  • the hPS cells may be cultured with a differentiation cocktail for about 5 days in order to differentiate the hPS cells into mesoderm.
  • the differentiation cocktail may optionally further comprise one or more of the following: FLT3L, TPO, IL-4 and IL-3.
  • the mesoderm cells may express one or more factors or markers expressed by mesoderm cells. For example increased expression of the mesoderm associated transcription factor, Brachyury, along with the decreased expression of hPS associated transcription factor Oct4 and Tra-160 may be indicative of the differentiation of hPS cells to mesoderm cells. Allowing the culture to continue to grow in the presence of the differentiation cocktail may facilitate further differentiation of the mesoderm cells, e.g. into cells of hematopoietic lineage.
  • the cell culture may be grown in the presence of the differentiation cocktail for a suitable length of time to differentiate the cells beyond mesoderm cells and into other hematopoietic lineage cells.
  • the cells may be grown at least about 6 days, at least about 7 days, at least about 8 days, at least about 9 days, at least about 10 days with the differentiation cocktail described herein thereby differentiating the hPS cells into hematopoietic stem cells.
  • the cells may express one or more markers expressed by hematopoietic stem cells. Suitable markers may include CD45, CD34, and HoxB4.
  • the cells may be grown at least about 22 days, at least about 23 days, at least about 24 days, at least about 25 days, at least about 26 days, at least about 27 days, at least about 28 days with the differentiation cocktail described herein thereby differentiating the hPS cells into monocytes.
  • the cells may express one or more markers expressed by monocytes.
  • Suitable markers may include CD 14, CD45 and CD 11 c.
  • the cells may be grown at least about 20 days, at least about 23, at least about 25 days, at least about 30 days, at least about 31 days, at least about 32 days, at least about 33 days, with the differentiation cocktail described herein thereby differentiating the hPS cells into imDC.
  • the invention provides a method of differentiating hPS cells in hematopoietic lineage cells comprising contacting the hPS cells with one or more differentiation cocktails such that the hPS cells differentiate into one or more hematopoietic lineage cell types.
  • the method may be comprised of multiple steps wherein one or more of the steps results in the differentiation of intermediate cell types of hematopoietic lineage.
  • the invention contemplates not only the execution of all of the steps set forth below, but also the execution of one or more individual steps in order to attain a desired intermediate or precursor cell type of hematopoietic lineage.
  • the mesoderm cells from above may be contacted with a second differentiation cocktail comprising VEGF, SCF, GM-CSF thereby differentiating the mesoderm cells into hematopoietic stem cells.
  • the cells may be cultured with this differentiation cocktail for about 1-5 days.
  • hematopoietic the stem cell may be further differentiated into a common myeloid progenitor (CMP) cell by contacting the hematopoietic stem cell with a differentiation cocktail comprising GMCSF.
  • the differentiation cocktail may further comprise SCF.
  • the cells may be cultured with this differentiation cocktail for about 1-10 days.
  • the CMP may be further differentiated into a common granulocytic/monocytic progenitor (GMP) cell by contacting the CMP with a third differentiation cocktail comprising GMCSF.
  • GMCSF granulocytic/monocytic progenitor
  • the cells may be cultured with this differentiation cocktail for about 1-5 days.
  • the GMP may be further differentiated into monocytes by contacting the GMP with a differentiation cocktail comprising GM-CSF.
  • the cells may be cultured with this differentiation cocktail for about 1-10 days.
  • the monocytes may be further differentiated into imDC by contacting the monocytes with a differentiation cocktail comprising GM-CSF and IL-4.
  • the cells may be cultured with this differentiation cocktail for about 1-5 days.
  • the imDC may be matured into mDC by contacting the imDC with any of the maturation cocktails described infra.
  • the cells may be cultured with the maturation cocktail from about 12-72 hours.
  • the cells may be cultured with the maturation cocktail for about 24 hours.
  • the cells may be cultured with the maturation cocktail for about 48 hours.
  • the time may be any value or subrange within the recited ranges, including endpoints.
  • the invention provides a method of differentiating hPS cells into imDC comprising contacting the hPS cells with a differentiation cocktail comprising the following: 1) BMP-4 ranging from about 10 ng/ml to about 75 ng/ml; and 2) GM-CSF ranging from about 25 ng/ml to about 75 ng/ml.
  • the concentration of each may be any value or subrange within the recited ranges, including endpoints.
  • the invention provides a method of differentiating hPS cells into imDC comprising contacting the hPS cells with a differentiation cocktail comprising the following: 1) BMP-4 ranging from about 10 ng/ml to about 75 ng/ml; 2) VEGF ranging from about 25 ng/ml to about 75 ng/ml; 3) SCF ranging from about 5 ng/ml to about 50 ng/ml; and 4) GM-CSF ranging from about 25ng/ml to about 75 ng/ml.
  • the concentration of each may be any value or subrange within the recited ranges, including endpoints.
  • the invention provides a method of enriching a myeloid progenitor cell population comprising isolating a CD45+ Hi population from a cell culture comprising a CD45+ Hi cell population and a CD45+ low cell population.
  • the invention provides a method of isolating a granulocyte progenitor cell comprising isolating a CD45+ low population from a cell culture comprising a CD45+ Hi cell population and a CD45+ low cell population. High and low are relative terms.
  • a CD45+ low cell population may refer to a cells having CD45 expression about 1-2 orders of magnitude above background
  • the CD45+ Hi cells may refer to cells having CD45 expression greater than 2 orders of magnitude above background as measured using any assay know in the art, e.g. immunofluorescence as measured using a fluorescence detector, e.g. Fluorescent Activated Cell Sorter (FACS).
  • FACS Fluorescent Activated Cell Sorter
  • Isolating the target cell population may be done using any means known in the art.
  • the cell populations may be isolated using a commercially available (FACS).
  • the cells may be isolated based on fluorescent intensity of a marker stained with a labeled ligand.
  • the labeled ligand may attach directly to the cell or indirectly to the cell by virtue of another ligand attached to the cell by the human hand.
  • the cell populations may be isolated based on size and density based on forward and side scatter on a cell sorter. As an example CD45+ Hi and CD45+ low populations may be separated using a cell sorter based on size and granularity.
  • the cytokine combinations useful in carrying out various embodiments of the invention may be used at any suitable final working concentration to achieve the desired effect.
  • BMP -4 may be used at a concentration ranging from about 1 ng/ml to about 120 ng/ml; from about 5 ng/ml to about 100 ng/ml; from about 10 ng/ml to about 80 ng/ml; from about 25 ng/ml to about 75 ng/ml; from about 30 ng/ml to about 60 ng/ml. In some embodiments of the invention about 50 ng/ml of BMP-4 may be used.
  • VEGF may be used at a concentration ranging from about 1 ng/ml to about 120 ng/ml; from about 5 ng/ml to about 100 ng/ml; from about 20 ng/ml to about 80 ng/ml; from about 25 ng/ml to about 75 ng/ml; from about 30 ng/ml to about 60 ng/ml. In some embodiments of the invention about 50 ng/ml of VEGF may be used.
  • GM- CSF may be used at a concentration ranging from about 1 ng/ml to about 120 ng/ml; from about 5 ng/ml to about 100 ng/ml; from about 20 ng/ml to about 80 ng/ml; from about 25 ng/ml to about 75 ng/ml; from about 30 ng/ml to about 60 ng/ml. In some embodiments of the invention about 50 ng/ml of GM-CSF may be used.
  • SCF may be used at a concentration ranging from about 1 ng/ml to about 350 ng/ml; from about 5 ng/ml to about 300 ng/ml; from about 10 ng/ml to about 250 ng/ml; from about 15 ng/ml to about 200 ng/ml; from about 20 ng/ml to about 150 ng/ml; from about 5 ng/ml to about 50 ng/ml. In some embodiments of the invention about 20 ng/ml of SCF may be used.
  • FLT3L may be used at a concentration ranging from about 1 ng/ml to about 350 ng/ml; from about 5 ng/ml to about 300 ng/ml; from about 10 ng/ml to about 250 ng/ml; from about 15 ng/ml to about 200 ng/ml; from about 20 ng/ml to about 150 ng/ml. In some embodiments of the invention about 20 ng/ml of FLT3L may be used.
  • IL-3 may be used at a concentration ranging from about 1 ng/ml to about 80 ng/ml; from about 5 ng/ml to about 75 ng/ml; from about 10 ng/ml to about 50 ng/ml; from about 20 ng/ml to about 40 ng/ml. In some embodiments of the invention about 25 ng/ml of IL-3 may be used.
  • TPO may be used at concentration ranging from about 1 ng/ml to about 150 ng/ml; from about 5 ng/ml to about 100 ng/ml; from about 10 ng/ml to about 80 ng/ml; from about 20 ng/ml to about 60 ng/ml.
  • IL-4 may be used at a concentration ranging from about 1 ng/ml to about 120 ng/ml; from about 5 ng/ml to about 100 ng/ml; from about 20 ng/ml to about 80 ng/ml; from about 25 ng/ml to about 75 ng/ml; from about 30 ng/ml to about 60 ng/ml. In some embodiments of the invention about 50 ng/ml of IL-4 may be used.
  • the concentration may be any value or subrange within the recited ranges, including endpoints.
  • a maturation cocktail comprising a plurality of cytokines may be used to mature imDC to mDC.
  • Suitable final working concentrations of cytokine components of the maturation cocktail may include any concentration which effectively matures imDC to mDC.
  • IFNy may be used at a concentration ranging from about 1 ng/ml to about 150 ng/ml; from about 5 ng/ml to about 100 ng/ml; from about 10 ng/ml to about 100 ng/ml; from about 15 ng/ml to about 80 ng/ml; from about 20 ng/ml to about 60 ng/ml. In some embodiments of the invention about 25 ng/ml of IFNy may be used.
  • TNFa may be used at a concentration ranging from about 1 ng/ml to about 200 ng/ml; from about 10 ng/ml to about 150 ng/ml; from about 20 ng/ml to about 100 ng/ml; from about 30 ng/ml to about 80 ng/ml; from about 40 ng/ml to about 75 ng/ml. In some embodiments of the invention about 10 ng/ml of TNFa may be used.
  • IL-Ib may be used at concentration ranging from about 1 ng/ml to about 200 ng/ml, from about 5 ng/ml to about 150 ng/ml; from about 8 ng/ml to about 75 ng/ml; from about 10 ng/ml to about 50 ng/m. In some embodiments of the invention about 10 ng/ml of IL-Ib may be used.
  • the concentration may be any value or subrange within the recited ranges, including endpoints.
  • PGE2 may be used at a concentration ranging from about 0.1 ug/ml to about 150 ug/ml; from about 0.5 ug/ml to about 100 ug/ml; from about 0.8 ug/ml to about 75 ug/ml; from about 1 ug/ml to about 50 ug/ml. In some embodiments of the invention about 1 ug/ml of PGE2 may be used.
  • Poly I:C may be used a concentration ranging from about 1 ug/ml to about 50 ug/ml, from about 5 ug/ml to about 40 ug/ml from about 10 ug/ml to about 30 ug/ml, form about 15 ug/ml to about 25 ug/ml. In some embodiments of the invention about 20 ug/ml of Poly I:C may be used.
  • the concentration may be any value or subrange within the recited ranges, including endpoints.
  • the invention provides for the differentiation of hPS cells in hematopoietic lineage cells wherein at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99% of cells express one or more markers or factors that are expressed by cells of hematopoietic lineage.
  • the concentration may be any value or subrange within the recited ranges, including endpoints.
  • Antigen presenting cells of this invention are often referred to in this disclosure as “dendritic cells”. However, this is not meant to imply any morphological, phenotypic, or functional feature beyond what is explicitly required.
  • the term is used to refer to cells that are phagocytic or can present antigen to T lymphocytes, falling within the general class of monocytes, macrophages, dendritic cells and the like, such as may be found circulating in the blood or lymph, or fixed in tissue sites. Phagocytic properties of a cell can be determined according to their ability to take up labeled antigen or small particulates. The ability of a cell to present antigen can be determined in a mixed lymphocyte reaction as described.
  • dendritic cells and antigen-presenting cells in the body are first identified in tissue sites such as the skin or the liver; but regardless of their origin, location, and developmental pathway, they are considered in the art to fall within the general category of hematopoietic cells.
  • tissue sites such as the skin or the liver; but regardless of their origin, location, and developmental pathway, they are considered in the art to fall within the general category of hematopoietic cells.
  • dendritic cells used in this disclosure also fall in the broad category of hematopoietic cells, whether produced through the hematopoietic or direct paradigm framed earlier, or through a related or combined pathway.
  • hPS derived cells as antigen-presenting cells is provided in this disclosure as an explanation to facilitate the understanding of the reader. However, the theories expostulated here are not intended to limit the invention beyond what is explicitly required.
  • the hPS derived cells of this invention may be used therapeutically regardless of their mode of action, as long as they achieve a desirable clinical benefit in a substantial proportion of patients treated.
  • antigen-presenting cells may include, but are not necessarily limited to, dendritic cells, macrophages, and B cells.
  • the antigen-presenting cells are dendritic cells.
  • the antigen expressing cells described herein express an antigen of an infectious agent.
  • the infectious agent is a virus, bacterium, parasite, protozoan, or a fungus.
  • the infection is caused by a virus.
  • the virus may be a DNA virus, a RNA virus, or a retrovirus.
  • viruses useful with the present invention include, but are not limited to Ebola, measles, SARS-CoV, SARS-CoV-2, Chikungunya, hepatitis, Marburg, yellow fever, MERS-CoV, Dengue, Lassa, influenza, rhabdovirus or HIV.
  • a hepatitis virus may include hepatitis A, hepatitis B, or hepatitis C.
  • An influenza virus may include, for example, influenza A or influenza B.
  • An HIV may include HIV 1 or HIV 2.
  • the viral sequence may be a human respiratory syncytial virus, Sudan ebola virus, Bundibugyo virus, Tai Forest ebola virus, Reston ebola virus, Achimota, Aedes flavivirus, Aguacate virus, Akabane virus, Alethinophid reptarenavirus, Allpahuayo mammarenavirus, Amapari mmarenavirus, Andes virus, acea virus, Aravan virus, Aroa virus, Arumwot virus, Atlantic salmon paramyoxivirus, Australian bat lyssavirus, Avian bomavirus, Avian metapneumovirus, Avian paramyoxviruses, penguin or Falkland Islandsvirus, BK polyomavirus, Bagaza virus, Banna virus, Bat hepevirus, Bat sapovirus, Bear Canon mammarenavirus, Beilong virus, Betacoronoavirus, Betapapillomavirus 1-6, Bhanja virus, Bo
  • Human papillomavirus Human parainfluenza virus 1-4, Human paraechovirus, Human picobimavirus, Human smacovirus, Ikoma lyssavirus, Ilheus virus, Influenza A-C, Ippy mammarenavirus, Irkut virus, J-virus, JC polyomavirus, Japanses encephalitis virus, Junin mammarenavirus, KI polyomavirus, Kadipiro virus, Kamiti River virus, Kedougou virus, Khujand virus, Kokobera virus, Kyasanur forest disease virus, Lagos bat virus, Langat virus, Lassa mammarenavirus, Latino mammarenavirus, Leopards Hill virus, Liao ning virus, Ljungan virus, Lloviu virus, Louping ill virus, Lujo mammarenavirus, Luna mammarenavirus, Lunk virus, Lymphocytic choriomeningitis mammarenavirus, Lyssa
  • Middle East respiratory syndrome coronavirus Mobala mammarenavirus, Modoc virus, Moijang virus, Mokolo virus, Monkeypox virus, Montana myotis leukoenchalitis virus, Mopeia lassa virus reassortant 29, Mopeia mammarenavirus, Morogoro virus, Mossman virus, Mumps virus, Murine pneumonia virus, Murray Valley encephalitis virus, Nariva virus, Newcastle disease virus, Nipah virus, Norwalk virus, Norway rat hepacivirus, Ntaya virus, O’nyong-nyong virus, Oliveros mammarenavirus, Omsk hemorrhagic fever virus, Oropouche virus, Parainfluenza virus 5, Parana mammarenavirus, Parramatta River virus, Peste-des-petits-ruminants virus, Pichande mammarenavirus, Picomaviridae virus, Pirital mammarenavirus, Piscihepevirus A, Procine parainfluen
  • RNA viruses that may be detected include one or more of (or any combination of) Coronaviridae virus, a Picornaviridae virus, a Caliciviridae virus, a Flaviviridae virus, a Togaviridae virus, a Bomaviridae, a Filoviridae, a Paramyxoviridae, a Pneumoviridae, a Rhabdoviridae, an Arenaviridae, a Bunyaviridae, an Orthomyxoviridae, or a Deltavirus.
  • the virus is Coronavirus, SARS-Coronavirus, Poliovirus, Rhinovirus, Hepatitis A, Norwalk virus, Yellow fever virus, West Nile virus, Hepatitis C virus, Dengue fever virus, Zika virus, Rubella virus, Ross River virus, Sindbis virus, Chikungunya virus, Borna disease virus, Ebola virus, Marburg virus, Measles virus, Mumps virus, Nipah virus, Hendra virus, Newcastle disease virus, Human respiratory syncytial virus, Rabies virus, Lassa virus, Hantavirus, Crimean-Congo hemorrhagic fever virus, Influenza, or Hepatitis D virus.
  • the infection is caused by a bacterium.
  • Non-limiting examples of bacteria that can be useful in accordance with the disclosed methods include without limitation any one or more of (or any combination of) Acinetobacter baumanii, Actinobacillus sp., Actinomycetes, Actinomyces sp. (such as Actinomyces israelii and Actinomyces naeslundii), Aeromonas sp.
  • Anaplasma phagocytophilum Anaplasma marginale Alcaligenes xylosoxidans, Acinetobacter baumanii, Actinobacillus actinomycetemcomitans, Bacillus sp. (such as Bacillus anthracis, Bacillus cereus, Bacillus subtilis, Bacillus thuringiensis, and Bacillus stearothermophilus), Bacteroides sp. (such as Bacteroides fragilis), Bartonella sp.
  • Bordetella sp. such as Bordetella pertussis, Bordetella parapertussis, and Bordetella bronchi septica
  • Borrelia sp. such as Borrelia recurrentis, and Borrelia burgdorferi
  • Brucella sp. such as Brucella abortus, Brucella canis, Brucella melintensis and Brucella suis
  • Burkholderia sp. such as Burkholderia pseudomallei and Burkholderia cepacia
  • Capnocytophaga sp. Cardiobacterium hominis, Chlamydia trachomatis, Chlamydophila pneumoniae, Chlamydophila psittaci, Citrobacter sp. Coxiella burnetii, Corynebacterium sp. (such as, Corynebacterium diphtheriae, Corynebacterium jeikeum and Corynebacterium), Clostridium sp.
  • Enterobacter sp such as Clostridium perfringens, Clostridium difficile, Clostridium botulinum and Clostridium tetani
  • Eikenella corrodens Enterobacter sp.
  • Enterobacter aerogenes such as Enterobacter aerogenes, Enterobacter agglomerans, Enterobacter cloacae and Escherichia coli, including opportunistic Escherichia coli, such as enterotoxigenic E. coli, enteroinvasive E. coli, enteropathogenic E. coli, enterohemorrhagic E. coli, enteroaggregative E. coli and uropathogenic E. coli
  • Enterococcus sp such as Clostridium perfringens, Clostridium difficile, Clostridium botulinum and Clostridium tetani
  • Eikenella corrodens Enterobacter sp.
  • Enterobacter aerogenes such as Enterobacter
  • Ehrlichia sp. (such as Enterococcus faecalis and Enterococcus faecium) Ehrlichia sp. (such as Ehrlichia chafeensia and Ehrlichia canis), Epidermophyton floccosum, Erysipelothrix rhusiopathiae, Eubacterium sp., Francisella tularensis, Fusobacterium nucleatum, Gardnerella vaginalis, Gemella morbillorum, Haemophilus sp.
  • Haemophilus influenzae such as Haemophilus influenzae, Haemophilus ducreyi, Haemophilus aegyptius, Haemophilus parainfluenzae, Haemophilus haemolyticus and Haemophilus parahaemolyticus
  • Helicobacter sp such as Helicobacter pylori, Helicobacter cinaedi and Helicobacter fennelliae
  • Kingella kingii Klebsiella sp.
  • Lactobacillus sp. Listeria monocytogenes, Leptospira interrogans, Legionella pneumophila, Leptospira interrogans, Peptostreptococcus sp., Mannheimia hemolytica, Microsporum canis, Moraxella catarrhalis, Morganella sp., Mobiluncus sp., Micrococcus sp., Mycobacterium sp.
  • Mycobacterium leprae such as Mycobacterium leprae, Mycobacterium tuberculosis, Mycobacterium paratuberculosis, Mycobacterium intracellulare, Mycobacterium avium, Mycobacterium bovis, and Mycobacterium marinum
  • Mycoplasm sp. such as Mycoplasma pneumoniae, Mycoplasma hominis, and Mycoplasma genitalium
  • Nocardia sp. such as Nocardia asteroides, Nocardia cyriacigeorgica and Nocardia brasiliensis
  • Neisseria sp such as Neisseria sp.
  • Prevotella sp. Porphyromonas sp., Prevotella melaninogenica, Proteus sp. (such as Proteus vulgaris and Proteus mirabilis), Providencia sp.
  • Rhodococcus sp. Rhodococcus sp.
  • Serratia marcescens Stenotrophomonas maltophilia
  • Salmonella sp. such as Salmonella enterica, Salmonella typhi, Salmonella paratyphi, Salmonella enteritidis, Salmonella cholerasuis and Salmonella typhimurium
  • Shigella sp. such as Shigella dysenteriae, Shigella flexneri, Shigella boydii and Shigella sonnei
  • Staphylococcus sp. such as Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus hemolyticus, Staphylococcus saprophyticus
  • Streptococcus sp such as Serratia marcesans and Serratia liquifaciens
  • Shigella sp. such as Shigella dysenteriae, Shigella flexneri, Shigella boydii and Shigella sonnei
  • Staphylococcus sp. such as Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus hemolyticus, Staphylococcus saprophyticus
  • Streptococcus pneumoniae for example chloramphenicol-resistant serotype 4 Streptococcus pneumoniae, spectinomycin-resistant serotype 6B Streptococcus pneumoniae, streptomycin-resistant serotype 9V Streptococcus pneumoniae, erythromycin-resistant serotype 14 Streptococcus pneumoniae, optochin-resistant serotype 14 Streptococcus pneumoniae, rifampicin-resistant serotype 18C Streptococcus pneumoniae, tetracycline-resistant serotype 19F Streptococcus pneumoniae, penicillin-resistant serotype 19F Streptococcus pneumoniae, and trimethoprim-resistant serotype 23F Streptococcus pneumoniae, chloramphenicol-resistant serotype 4 Streptococcus pneumoniae, spectinomycin-resistant serotype 6B Streptococcus pneumoniae, streptomycin-resistant serotype 9V Streptococcus pneumoniae, chlor
  • the infection is caused by a parasite.
  • parasites useful in accordance with disclosed methods include without limitation one or more of (or any combination of), an Onchocerca species and a Plasmodium species.
  • the infection is caused by a protozoan.
  • protozoa that can be useful in accordance with the disclosed methods and devices include without limitation any one or more of (or any combination of), Euglenozoa, Heterolobosea, Vaccinonadida, Amoebozoa, Blastocystic, and Apicomplexa.
  • Example Euglenoza include, but are not limited to, Trypanosoma cruzi (Chagas disease), T. brucei gambiense, T. brucei rhodesiense, Leishmania braziliensis, L. infantum, L. mexicana, L. major, L. tropica, and L. donovani.
  • Example Heterolobosea include, but are not limited to, Naegleria fowleri.
  • Example Vaccinona did include, but are not limited to, Giardia intestinalis (G. lamblia, G. duodenalis).
  • Example Amoebozoa include, but are not limited to, Acanthamoeba castellanii, Balamuthia madrillaris, Entamoeba histolytica.
  • Example Blastocystis include, but are not limited to, Blastocystic hominis.
  • Example Apicomplexa include, but are not limited to, Babesia microti, Cryptosporidium parvum, Cyclospora cayetanensis, Plasmodium falciparum, P.
  • the infection is caused by a fungus.
  • fungi examples include without limitation any one or more of (or any combination of), Aspergillus, Blastomyces, Candidiasis, Coccidiodomycosis, Cryptococcus neoformans, Cryptococcus gatti, sp. Histoplasma sp. (such as Histoplasma capsulatum), Pneumocystis sp. (such as Pneumocystis jirovecii), Stachybotrys (such as Stachybotrys chartarum), Mucroymcosis, Sporothrix, fungal eye infections ringworm, Exserohilum, Cladosporium.
  • Aspergillus Blastomyces, Candidiasis, Coccidiodomycosis, Cryptococcus neoformans, Cryptococcus gatti, sp. Histoplasma sp. (such as Histoplasma capsulatum), Pneumocystis s
  • the fungus is a yeast.
  • yeast that can be detected in accordance with disclosed methods include without limitation one or more of (or any combination of), Aspergillus species (such as Aspergillus fumigatus, Aspergillus flavus and Aspergillus clavatus), Cryptococcus sp.
  • the fungi is a mold.
  • Example molds include, but are not limited to, a Penicillium species, a Cladosporium species, a Byssochlamys species, or a combination thereof.
  • the antigen-expressing cells express an antigen of a Coronaviridae virus.
  • the Coronaviridae virus is SARS-CoV-2.
  • the Coronaviridae antigen comprises the Coronaviridae spike (S) protein.
  • the Coronaviridae antigen comprises the full-length, 1,273 amino acid spike protein.
  • the Coronaviridae antigen comprises the spike protein receptor-binding domain (RBD).
  • the Coronaviridae antigen comprises a 209 amino acid sequence of the RBD.
  • the Coronaviridae antigen comprises the spike protein SI domain, S2 domain, or both.
  • the Coronaviridae antigen comprises the nucleocapsid protein.
  • the Coronaviridae antigen comprises the membrane protein.
  • genetically altered As used herein, “genetically altered”, “transfected”, or “genetically transformed” refer to a process where a polynucleotide has been transferred into a cell by any suitable means of artificial manipulation, or where the cell is a progeny of the originally altered cell and has inherited the polynucleotide.
  • the polynucleotide will often comprise a transcribable sequence encoding a protein of interest, which enables the cell to express the protein at an elevated level or may comprise a sequence encoding a molecule such as siRNA or antisense RNA that affects the expression of a protein (either expressed by the unmodified cell or as the result of the introduction of another polynucleotide sequence) without itself encoding a protein.
  • the genetic alteration is said to be "inheritable” if progeny of the altered cell have the same alteration.
  • the cells of this invention can also be genetically altered in order to enhance their ability to be involved in modulating an immune response, or to deliver a therapeutic gene to a site of administration.
  • a vector is designed using the known encoding sequence for the desired gene, operatively linked to a promoter that is either pan-specific or specifically active in the differentiated cell type.
  • the promoter may be an inducible promoter that permits for the timed expression of the genetic alteration.
  • the cells may be genetically engineered to express a cytokine that modulates an immune response either by enhancing the response or dampening the response.
  • the cells are permanently transduced with a gene that enables the cells to express the gene product in progeny that bear characteristics of dendritic cells.
  • the cells can be transduced while they are still undifferentiated hPS cells, or at an intermediate stage (such as a hematopoietic or dendritic cell precursor).
  • Methods for genetically altering hPS cells in the presence or absence of feeder cells using lipofectamine are described in US 2002/0168766 A1 (Geron Corp.). Other transfections methods, including electroporation, may be used. Lentiviral and retroviral vectors are also suitable.
  • the expression cassette can be placed into a known location in the genome of the cell by homologous recombination (US 2003/0068818 Al).
  • cytokines such as IL-12 or IL-15 that contribute to cytotoxic T cell activation or memory
  • chemokine equivalents such as secondary lymphoid tissue chemokine (SLC), IFNy (which induces monokine), or lymphotactin (Lptn).
  • SLC secondary lymphoid tissue chemokine
  • IFNy which induces monokine
  • Lptn lymphotactin
  • the hPS cells are genetically altered before they are differentiated.
  • the hPS cells are genetically altered after they are differentiated.
  • the hPS are genetically altered by transfecting them with a polynucleotide encoding a Coronaviridae virus antigen and the cytoplasmic tail of lysosomal associated membrane protein 1 (LAMP-1).
  • the polynucleotide further comprises a poly(A) tail and a gene which encodes heat shock protein 96 (HSP96), as described in the examples and in Figure 12.
  • the dendritic cell preparations described in this disclosure are formulated for administration to a human subject. This means that the cells are prepared in compliance with local regulatory requirements, are sufficiently free of contaminants and pathogens for human administration, and are suspended in isotonic saline or other suitable pharmaceutical excipient.
  • mDC may be administered to a human subject to stimulate an immune response in the subject.
  • the imDC Prior to administration, the imDC may be contacted with an antigen of interest and then matured into mDC.
  • the antigen may be internalized and processed such that it is presented on the cell surface in the context of MHC I and/or MHC II and thus may stimulate a specific immune response to the antigen.
  • the specific immune response may have a therapeutic effect.
  • the immune response may provide a prophylactic effect.
  • the specific immune response may provide a source of antigen specific cells such as cytotoxic T cells, or B lymphocytes or antibodies which specifically recognize the antigen.
  • Administration of the cells according to the invention may be by intravenous, intradermal or intramuscular injection. In other embodiments the cells may be administered subcutaneously.
  • the cells may be formulated with an appropriate buffer, such as PBS and/or an appropriate excipient.
  • the cells may be formulated with a suitable adjuvant. Examples of suitable pharmaceutical carriers are described in Remington's Pharmaceutical Sciences by E.W. Martin 20th Edition. Baltimore, MD: Lippincott Williams & Wilkins, 2000. [0090] For general principles in medicinal formulation and use of cellular vaccine compositions, the reader is referred to Handbook of Cancer Vaccines by M.A. Morse et ah, Humana Press, 2004; Cancer Vaccines and Immunotherapy by P.L.
  • any of the dendritic cell preparations of this invention can be stored after preparation to be used later for therapeutic administration or further processing.
  • Methods of cryoconserving dendritic cells both before and after loading are described in PCT publication WO 02/16560 (B. Schuler-Thumer et al.).
  • Occasional reference to a pharmaceutical composition in this disclosure as a “vaccine” implies no particular mode of action or administration. The term means only that it has been formulated for administration to a human subject as already described.
  • a vaccine may be designed as an immunogenic composition for generating a CTL response against a target antigen — but this need not be demonstrated as long as the composition is therapeutically effective according to any suitable clinical criterion in a reasonable proportion of treated cancer patients.
  • Various cell preparations of this invention can be maintained or supplied in combination with each other or with materials useful in their manufacture or use.
  • hPS derived dendritic cells include any system or combination of cells or reagents that exist at any time during manufacture, distribution, testing, or clinical use of the hPS derived dendritic cells, as described in this disclosure.
  • Cell populations that may be useful together are undifferentiated hPS cells, hPS-derived dendritic cell precursors, mature dendritic cells, toleragenic dendritic cells, or other differentiated cell types, in any combination, sometimes derived from the same hPS cell line.
  • Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions which can also contain buffers, diluents and other suitable additives.
  • non-aqueous solvents examples include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate.
  • Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media.
  • Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils.
  • Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives can also be present such as, for example, antimicrobials, anti-oxidants, chelating agents, and inert gases and the like.
  • Formulations for topical administration can include ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders.
  • Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like can be necessary or desirable.
  • compositions for oral administration can include powders or granules, suspensions or solutions in water or non-aqueous media, capsules, sachets, or tablets. Thickeners, flavorings, diluents, emulsifiers, dispersing aids or binders can be desirable.
  • various cell preparations described herein can be maintained or supplied in combination with each other or with materials useful in their manufacture or use.
  • Commercial embodiments include any system or combination of cells or reagents that exist at any time during manufacture, distribution, testing, or clinical use of the hPS derived dendritic cells, as described in this disclosure.
  • Cell populations that may be useful together are undifferentiated hPS cells, hPS-derived dendritic cell precursors, mature dendritic cells, toleragenic dendritic cells, or other differentiated cell types, in any combination, sometimes derived from the same hPS cell line.
  • Pharmaceutical compositions of this invention may optionally be packaged in a suitable container with written instructions for a desired purpose, such as the reconstitution of hematopoietic-lineage cell function to improve a disease condition or to stimulate an immune response.
  • a compound or pharmaceutical composition described herein can be administered to the subject in a number of ways depending on whether local or systemic treatment is desired, and on the area to be treated.
  • a compound or pharmaceutical composition described herein can be administered as an ophthalmic solution and/or ointment to the surface of the eye.
  • a compound or pharmaceutical composition can be administered to a subject vaginally, rectally, intranasally, orally, by inhalation, or parenterally, for example, by intradermal, subcutaneous, intramuscular, intraperitoneal, intrarectal, intraarterial, intralymphatic, intravenous, intrathecal and intratracheal routes. Parenteral administration, if used, is generally characterized by injection.
  • Injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution or suspension in liquid prior to injection, or as emulsions.
  • a more recently revised approach for parenteral administration involves use of a slow release or sustained release system such that a constant dosage is maintained. See, e.g., U.S. Pat. No. 3,610,795, which is incorporated by reference herein.
  • the methods provided herein include a composition for treating an infectious disease.
  • the composition may comprise a formulation of differentiated hPS cells as described elsewhere herein.
  • the hPS may be differentiated into a population of mature antigen- presenting cells, wherein said antigen expressing cells express an antigen of an infectious agent, as described elsewhere herein.
  • the differentiated hPS cells may also be genetically altered before or after they are differentiated so that they express a protein comprising one or more immunogenic epitopes of the infectious agent.
  • the composition may then be administered to a human for preventing an infectious disease.
  • the cellular compositions are made from stem cells, particularly pluripotent stem cells of human origin.
  • the culture is differentiated into cells having characteristics of antigen-presenting cells, loaded with a specific target antigen on an infectious particle, and formulated for administration to a human subject.
  • the differentiation process involves culturing the cells in an environment of cytokines and other factors that generate a hematopoietic or early dendritic cell progenitor, and then maturing the cells to the phenotype intended for administration. Effective factor combinations and markers to effect and monitor the differentiation procedure are provided elsewhere in the disclosure.
  • the antigen-presenting cells are selected from the group consisting of dendritic cells, macrophages, B cells, and a combination thereof.
  • the antigen-presenting cells are dendritic cells.
  • the infectious disease is a viral infection caused by a virus.
  • the antigen-presenting cells express an antigen of said virus.
  • the virus is a Coronaviridae virus. In specific embodiments, the Coronaviridae virus is SARS-CoV-2.
  • the composition is administered to a human for treating an infectious disease.
  • the composition is administered to a human for preventing an infectious disease.
  • the composition is a vaccine.
  • the disclosure also provides a composition comprising dendritic cells expressing a Coronaviridae antigen.
  • the Coronaviridae antigen comprises the Coronaviridae spike protein.
  • the composition comprises the whole, 1,273 amino acid spike protein.
  • the composition comprises the spike protein receptor-binding domain (RBD).
  • the Coronaviridae antigen comprises a 209 amino acid sequence of the RBD.
  • the Coronaviridae antigen comprises the spike protein SI domain, S2 domain, or both.
  • the composition comprises the Coronaviridae nucleocapsid protein.
  • the composition comprises the Coronaviridae membrane protein.
  • Example 1 Engineer DC Cells with a Relevant SARS-CoV-2 Antigen
  • the workhorse tool to modulate or prepare an immune system to fight aggressing pathogens is the vaccine. This approach consists of presenting antigens from pathogens to our immune system such that we prepare defense mechanisms ahead of a future infection to prevent it from taking hold in our body.
  • the vaccine development timeline is often outpaced by the propagation of emerging diseases, adding substantial pressure to healthcare systems. This effectively limits their utilities in the early phase of disease propagation.
  • Adoptive immunotherapy is a novel type of therapeutic approach that has shown tremendous potential in fighting cancer.
  • Adoptive immunotherapy generally consists of engineering the effector cells of our immune system (DCs and T Cells) to respond by detecting and destroying cancer cells.
  • DCs and T Cells immune system
  • cancer vaccines Similar to traditional vaccines, one limitation of cancer vaccines is that it usually takes time to select a relevant target antigen to fight a specific disease and engineer large cell banks to address broad segment populations.
  • Competing approaches rely mostly on engineering a patient’s own immune cells such at T Cells as a first step, followed by expansion and reinfusion of the modified cells within the same patient.
  • Applicants’ strategy is built on its ability to produce large cell banks from pluripotent universal donor cells that are terminally differentiated into DCs, the main APC of the immune system. Such cell banks can then be modified with a relevant antigen shortly prior to injecting patients to elicit an immune response, a concept called a DC Vaccine. This fundamental difference could greatly accelerate development and improve accessibility for broad patient populations.
  • Such a “plug-and-play” approach can be tailored initially to express SARS-CoV-2 relevant antigens to treat COVID-19, while large DC banks can be stored and modified quickly with future emerging diseases antigens. Table 1.
  • Phase I DC cells will be engineered with a relevant SARS-CoV-2 antigen such as the Spike protein for downstream testing in a small clinical study.
  • a relevant SARS-CoV-2 antigen such as the Spike protein for downstream testing in a small clinical study.
  • Applicants already benefit from fully characterized human pluripotent cell banks, robust process development, quality assurance and regulatory teams, and validated clean rooms for cGMP production.
  • Phase II In parallel to preclinical testing requirements necessary to initiate human trials, Applicants will work on scale up processes to establish methods and pathways to bring such therapy to large scale population. This will demonstrate an ability to address future large scale needs for emerging threats.
  • DC vaccines offer a unique opportunity for a “One- Two-Punch” for COVID-19 and other emerging diseases.
  • DCs can act not only on activation of killer T cells and macrophages to eliminate ongoing infections, but also could play a role in the preservation of an immune memory for future infections.
  • trials will be designed in both diseased and naive patient population.
  • a key technical risk resides on the effectiveness of any given antigen such as the Spike protein of SARS-CoV-2 to activate a relevant immune response.
  • use of a combination of antigens might lead to an improved therapeutic approach.
  • the platform described herein allows for the simultaneous introduction of various antigens within the same batch of DC for multiple target presentation.
  • Example 2 Dendritic cell vaccines for cancer treatment and COVID-19 prevention
  • the Dendritic Cell Infectious Disease Program builds on the previously described VAC Platform using mature dendritic cells to increase a patient’s immune response.
  • the dendritic cell vaccine is an allogenic (“off the shelf’) vaccine.
  • Cells are manufactured from a pluripotent cell line and not derived from the patient. This provides advantages in terms of time and cost. Mature dendritic cells are manufactured and loaded with relevant antigen to stimulate CD8+ (cytotoxic) and CD4+ (helper) T cell responses. “Targeted education” of T cells increases immune response and pathogen destruction (Figure 1).
  • Figure 2 highlights results from the Phase II, multicenter, open-label trial. 19 patients with acute myeloid leukemia (AML) were treated and they ended up in complete remission. These were intermediate and high risk patients, evaluated by cytogenetics. Seven of these treated patients were in a very high risk group, over the age of 60. The treatment showed robust safety in all patients treated, with a clear anti-telomerase immune response.
  • AML acute myeloid leukemia
  • the VAC1 program involved autologous dendritic cell therapy for AML.
  • hTERT- specific T cell responses were induced in 58% of patients. 58% remained in cancer remission at the median duration of follow-up of 52 months from first vaccination.
  • the response was reproducible: 3 of 4 patients exhibited increased hTERT-specific T cells by pentamer assay that coincided with their dendritic cell vaccination schedule.
  • the percentage of hTERT-specific T cells detected by pentamer staining ranged from 0.75% to 3.78% in patients treated to date, similar to other approved cancer vaccines.
  • the frequency of antigen-specific T cells required for immunity against infectious diseases can be as low as 0.1% (typically 0.2-0.6%) of the total T cell population.
  • the magnitude of the hTERT-specific T cell response generated by VAC2 is viewed as being of potential biological relevance and warrants further investigation. There is also evidence of durability: for the two VAC2 patients with pre- and post vaccination pentamer assay data, the apparent sustained increase in hTERT-specific T cells provides evidence of a durable immune response. [0127] Dendritic cell vaccines allow for rapid deployment to fight the COVID-19 pandemic and prepare for future emerging threats.
  • a dendritic cell bank and arsenal can be built now to [0128] Produce cGMP grade fully tested pluripotent cell banks. In house development and manufacturing operations allow for large scale differentiated dendritic cells (drug substance). Ongoing and emerging threats can then be monitored for relevant antigens.
  • dendritic cells can be armed with the relevant antigen.
  • the cGMP antigen can be generated - any type of antigen is possible, including peptides, protein, oligonucleotides, or mRNA.
  • the antigen can be electroporated into the dendritic cells, and stocks can be cryopreserved and deployed to repositories.
  • Dendritic cell vaccines are formulated and cryopreserved for simple thawing and injection.
  • the vaccines can be distributed and administered.
  • the ready to use vaccines can be sent to repositories and treatment centers, stored frozen until use, then thawed, loaded into syringes and administered by intradermal injection.
  • dendritic cell therapy is a unique opportunity to complement ongoing efforts to fight COVID-19.
  • the availability of cGMP cell banks permits rapid development of clinical grade material for human testing.
  • the availability of relevant (or suspected) antigen targets can be uploaded quickly into naive dendritic cells.
  • the safety profile in humans has been established in cancer immunotherapy clinical trials.
  • the pathway from clinical material availability to human trials is fast - it only requires an in vitro bioassay because efficacy testing in animals is not possible.
  • This system could provide tools to both fight active COVID-19 infection and provide multi-year memory immunity. It is broadly applicable to any infectious disease.
  • This product could play a critical role in certain settings, genetic or professional, due to its differentiated approach to both treating disease and providing lasting immunity.
  • Example 3 Expression of mRNA in dendritic cells.
  • dendritic cells were derived from monocytes extracted from peripheral blood mononuclear cells, which were collected and cultured in a manner that enriches for antigen-presenting cells. Purified dendritic cells were then electroporated with mRNA coding for GFP or SARS-CoV-2 Spike protein. Expression of the spike protein was assessed 16 hours post electroporation by intracellular staining of CD83, CD86, HLA-DR, followed by flow cytometry. Percent expression of the spike and GFP proteins is shown in Table 2. Monocytes were differentiated to mature dendritic cells as expected showing high expression of CD83, CD86, and HLA-DR (>90%). CD83 is a marker for dendritic cell maturation, while CD86 and HLA-DR are costimulatory molecules involved in T cell activation.
  • Control (2) consisted of fresh mDCs that were not electroporated
  • control (4) consisted of fresh mDCs with spike mRNA that were not electroporated
  • A consisted of fresh mDCs with spike mRNA (Invitrogen) that were electroporated
  • B consisted of fresh mDCs with spike mRNA (NEB) that were electroporated.
  • Monocytes were cryopreserved in CS-10 and frozen in vapor phase of nitrogen for further use.
  • Monocytes were cultured on ultra low attachment T75 flasks (Corning) in CellGenix DC supplemented with GM-CSF and IL-4. Medium was replaced on day 1 and day 4.
  • monocytes were differentiated into immature dendritic cells (iDC) and were stimulated with CellGenix DC medium supplemented with GM-CSF, TNF-a, IFN-g, IL- 1b, and PGE2 for additional 24 hours for generation of mature dendritic cells.
  • iDC immature dendritic cells
  • Plasmid DNA SARS-CoV-2 construction and mRNA synthesis Plasmid DNA SARS-CoV-2 construction and mRNA synthesis. Plasmid DNA containing SARS-CoV-2 surface glycoprotein was constructed by cloning SARS-CoV-2 full length spike protein sequence (aal6-1212) into the pKAN-hTERT-LAMPl as a vector backbone where the hTERT sequence was removed and swapped with the SARS-CoV-2 spike protein sequence ( Figures 9 and 10).
  • DNA fragments that encode the NH2 -terminal signal peptide sequence (amino acids 1-27) of the heat shock protein 96 (gp96) and the endosomal/lysosomal targeting signal (amino acids 382-416) of LAMP- 1 were ligated into restriction sites present in the SARS-CoV-2 spike protein.
  • the SARS-CoV-2/COVID 19/LAMP-l mRNA was under the control of the bacteriophage T7 promoter.
  • mRNA was synthesized from the plasmid DNA using Invitrogen mRNA synthesis kit mMESSAGE mMACHINETM T7 Transcription Kit, Cat #AM1344 as well as HiScribe T7 ARCA mRNA kit (with tailing) Cat# E2060. Briefly:
  • Plasmid DNA was linearized using restriction enzyme Spel.
  • Linearized plasmid DNA was purified using 1/20* 11 volume of 0.5M EDTA, 1/10 th volume of 3M Na acetate and 2 volume of ethanol.
  • the plasmid DNA was resuspended in Tris EDTA buffer pH7.5-8.0. Reactions for mRNA synthesis were assembled at room temperature. For a 20ul volume, 2XNTP/CAP was added, as well as 10X reaction buffer, linearized template DNA and enzyme mix. The reaction was mixed thoroughly by gently flicking the tube and by short centrifugation. The reaction mix was incubated at 37°C for lhr. After 1 hr, TURBO DNase was added to the reaction mix to degrade the residual DNA at 37°C for 15 min. Synthesized mRNA was purified using MEGAclearTM transcription clean up kit. Briefly:
  • Synthesized mRNA was brought to 100 ul by adding Elution buffer, then added 350 ul binding solution and 250 ul 100% ethanol. Mixed gently by pipetting.
  • the reaction mix was passed through the filter cartridge and washed twice with wash solution.
  • the mRNA was eluted from the filter cartridge using 50ul elution buffer. Eluted mRNA was quantified using Nanodrop.
  • Eluted mRNA had a concentration of 2062.6 ng/ul and the purity ratio A260/280 was 2.35 and A260/230 was 2.63.
  • the size of the mRNA was confirmed by running the mRNA on the denaturing RNA agarose gel from LONZA.
  • the purified SARS-CoV-2 (COVID 19) spike protein/LAMP-1 mRNA size was 4kb, which includes a signal sequence 81bp + full length covid spike protein 3819 bp + Lampl 102 bp ( Figure 11).
  • Dendritic cell preparation for electroporation Dendritic cell preparation was done according to the method procedure protocol. Briefly:
  • HLA-DR For surface staining (HLA-DR, CD83 and CD86), cells were stained with primary conjugated antibody in FACS buffer followed by flow cytometry analysis.
  • Spike protein For intra-cellular staining (Spike protein)- cells were fixed and permeabilized with Foxp3 Fix/Perm kit followed by staining with primary antibody against Spike protein (0.4 ⁇ g/test) and staining with secondary antibody (Goat anti-Rabbit) at a 1:200 dilution.
  • Buffy coat derived monocytes were differentiated to mature DCs showing high expression of CD83, CD86 and HLA-DR biomarkers ( > 95%).
  • Monocyte-derived mature dendritic electroporated with eGFP mRNA were 60.7% positive for GFP expression 16 hours post electroporation.
  • Positive control HEK 293 T transiently transfected with lentivirus vector of SARS- CoV-2 Spike were 21.2% positive for spike protein.
  • Negative control HI hESC were 0.23% positive for spike protein.
  • Monocyte-derived mature dendritic cells not electroporated with SARS- CoV-2 Spike mRNA or incubated with SARS-CoV-2 Spike mRNA without electroporation were 1.72% or 1.06% positive for spike, respectively (baseline).
  • Monocyte-derived mature dendritic cells electroporated with SARS-CoV-2 Spike mRNA generated using the Invitrogen IVT kit were 5.81% positive for spike protein 16 hours post electroporation.
  • Monocyte-derived mature dendritic cells electroporated with SARS-CoV-2 Spike mRNA generated using the NEB IVT kit were 1.16% positive for spike protein (same as the baseline) 16 hours post electroporation.
  • Example 4 DNA construct for in vitro transcription.
  • a DNA construct for in vitro transcription is designed and optimized for antigen presentation.
  • the construct is based on that previously used in the VAC1 and VAC2 clinical trials which utilized hTERT as the antigen.
  • the mRNA encoded includes portions of the Spike sequence and other SARS-CoV-2 sequences yet to be determined and the cytoplasmic tail of lysosomal associated membrane protein 1 (LAMP-1), which facilitates human leukocyte antigen HLA II, as well as HLA I loading of target protein. Both elements are fused to the sequence encoding heat shock protein 96 (HSP96) to ensure that the protein translated from the mRNA construct is efficiently shuttled to the endoplasmic reticulum and subsequently expressed on the cell surface.
  • Two candidate mRNA sequences are planned based on literature and other ongoing vaccine programs: Sars-COV-2S (RBD domain 209 aa sequence ID 209 aa and Sars-COV-2S (1,273 aa) ( Figure 12).
  • antigen/LAMP chimeras were found to elicit a much greater immune response than wild-type antigen.
  • This approach has proved useful in increasing cellular and humoral responses to several virus antigens, including human papillomavirus E7, dengue virus membrane protein, hepatitis C virus NS3 protein and cytomegalovirus pp65 (see, e.g., Bonini, et al., J. Immunol. 166:5250-5257; 2001).
  • the enhanced immune response can be attributed to co-localization of LAMP with MHC II and the more efficient processing and delivery of antigenic peptides.
  • LAMP -targeting is reported to result in the presentation of an increased number of immunogenic epitopes, thus inducing a qualitatively broadened immune response compared to untargeted antigen.
  • Fernandes et al. demonstrated an increase in the number of presented peptides of a LAMP- trafficked OVA antigen encoded in a vaccinia vector.
  • 9 were presented by an OVA/LAMP chimera, as compared to only 2 by the construct without LAMP.

Abstract

L'invention concerne des méthodes et des compositions pour l'ingénierie de cellules présentatrices d'antigène (CPA), telles que des cellules dendritiques (DC), des macrophages et des lymphocytes B, qui sont modifiées pour exprimer des antigènes viraux. À l'aide de cette approche, de grandes banques de cellules peuvent être créées et rapidement modifiées et déployées pour lutter contre divers types de maladies infectieuses telles que celles associées à des coronavirus et à d'autres infections virales.
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